Asthma has become a serious public health issue worldwide, and its prevalence has doubled in numerous industrialized countries (Beasley et al., J Allergy Clin Immunol 2000; 105, S466-472). Chronic airway inflammation characterized by pathological immune response is considered the hallmark of asthma. The key features of asthma include the production of allergen-specific IgE, resulting in immediate-type hypersensitivity reactions followed by the development of late phase responses such as eosinophil recruitment, mucus production, and airway hyperresponsiveness (AHR). In addition, eosinophilic inflammation driven by Th2-cytokines (including IL-4, IL-5 and IL-13) is deemed to play a crucial role in the pathogenesis of asthma (Renauld et al., J Clin Pathol 2001; 481:54, 577-589; Hoshino et al., Int Immunol 2004; 16, 1497-1505). Particularly, several studies have highlighted the crucial contribution of IL-13 in promoting the development of asthmatic features.
Acidic mammalian chitinase (AMCase), the prototypic chitinase, has been found to be induced during Th2-mediated inflammation through an IL-13-dependent mechanism (Kawada et al., Keio J Med 2007; 56, 21-27). It is a 50-kDa protein, containing a 39-kDa N-terminal catalytic domain that hydrolyzed chitin, a hinge region, and a C-terminal chitin-binding domain. AMCase is highly expressed in the lungs of asthmatic patients, as well as in mice models of asthma (Ramanathan et al., Am J Rhinol 2006; 20:479, 330-335). In fact, the hyper-expression of AMCase has also been found in the other airway tissues including the alveolar macrophages and lung epithelial cells in OVA-stimulated mice (Zhu et al., Science 2004; 304, 1678-1682). Inhibition of AMCase activity with specific antibodies appeared to be able to reduce the inflammatory response in BALF and lung tissues. However, neutralization of AMCase activity does not directly affect the expression of IL-4 and IL-13 (Zhu et al., Science 2004; 304, 1678-1682). It was found that AMCase could modulate the expression of several proinflammatory chemokines-including macrophage inflammatory protein (MIP)-1β, macrophage chemoattractant protein (MCP)-1 and eotaxin that play a crucial role in Th2-mediated airway inflammation (Mori, et al. Int Arch Allergy Immunol 2006; 140 Suppl 1, 55-58).
RNA interference (Fire, et al. Nature 1998; 391, 806-811) has become a powerful tool in downregulation of gene expression in mammalian cells and animal models (Cullen, et al. Gene Ther 2004; 13, 503-508). Recent studies have shown that short interfering (21-25 bp) RNA molecules (siRNA—small interfering RNA), but not long dsRNA (greater than 30 bp), are key elements of RNAi and appear to inhibit gene expression. Short hairpin RNA (shRNA) has been shown to be efficiently processed into siRNA inside the cells. In the last few years, some methods for expressing siRNAs in cells have been developed based on transcription of short hairpin RNAs (shRNAs) by RNA polymerase III promoter (Sui et al., Proc Natl Acad Sci USA 2002; 99, 5515-5520), such as U6 and H1. Delivery of siRNA into mammalian cells has been achieved via liposome, polymer and viral vectors. (Moore et al., J Gene Med 2005; 7, 918-925; Urban-Klein et al., Gene Ther 2005; 12, 461-466; Xu et al., Mol Ther 2005; 11, 523-530; Li et al., Cell Cycle; 5, 2103-2109; Aigner et al., Curr Opin Mol Ther 2007; 9, 345-352). Viral vectors appear to have the highest delivery efficiency. A serious problem with viral vectors is their immunogenity. In this regard, repeated applications may result in the production of neutralizing antibodies by the host. To overcome this issue, the use of adeno-associated virus (AAV), which promotes longterm transgene expression, has been proposed. AAV vector offers a compromise between an adequate level of transduction and an acceptable safety profile (Leung et al., J Gene Med 2007; 9, 10-21). Hence, several reports have successfully used AAV-mediated shRNA therapeutic system in controlling viral infections (Ge et al., Proc Natl Acad Sci USA 2004; 101, 8676-8681) and genetic disorders Rodriguez-Lebron et al., Mol Ther 2005; 12, 618-633). In the present study, we demonstrated that specific suppression of elevated AMCase results in a reduced eosinophilic and Th2-mediated airway inflammation in a mouse model of asthma. We also investigated whether the inhibition of AMCase may be associated with a reduced expression of IL-13, eotaxin, and other proinflammatory molecules.